Starch is the major form of carbon reserve in plants. constituting 50% or more of the dry weight of many storage organs--e.g. tubers. seeds of cereals. Starch is used in numerous food and industrial applications. In many cases, however. it is necessary to modify the native starches, via chemical or physical means. in order to produce distinct properties to suit particular applications. It would be highly desirable to be able to produce starches with the required properties directly in the plant, thereby removing the need for additional modification. To achieve this via genetic engineering requires knowledge of the metabolic pathway of starch biosynthesis. This includes characterisation of genes and encoded gene products which catalvse the synthesis of starch. Knowledge about the regulation of starch biosynthesis raises the possibility of re-programming biosynthetic pathways to create starches with novel properties that could have new commercial applications.
The commercially useful properties of starch derive from the ability of the native granular form to swell and absorb water upon suitable treatment. Usually heat is required to cause granules to swell in a process known as gelatinisation, which has been defined (W. A. Atwell et al., Cereal Foods World 33, 306-311. 1988) as " . . . the collapse (disruption) of molecular orders within the starch granule manifested in irreversible changes in properties such as granular swelling, native crvstallite melting, loss of birefringence. and starch solubilisation. The point of initial gelatinisation and the range over which it occurs is governed by starch concentration, method of observation, granule type, and heterogeneities within the granule population under observation". A number of techniques are available for the determination of gelatinisation as induced by heating, a convenient and accurate method being differential scanning calorimetry, which detects the temperature range and enthalpy associated with the collapse of molecular orders within the granule. To obtain accurate and meaningful results, the peak temperature of the endotherm observed by differential scanning calorimetry is usually determined.
The consequence of the collapse of molecular orders within starch granules is that the granules are capable of taking up water in a process known as pasting, which has been defined (W. A. Atwell et al., Cereal Foods World 33, 306-311, 1988) as ". . . the phenomenon following gelatinisation in the dissolution of starch. It involves granular swelling, exudation of molecular components from the granule. and eventually. total disruption of the granules". The best method of evaluating pasting properties is considered to be the viscoamylograph (Atwell et al., 1988) in which the viscosity of a stirred starch suspension is monitored under a defined time/temperature regime. A typical viscoamylograph profile for potato starch is shown in FIG. 5, in which the initial rise in viscosity is considered to be due to granule swelling. At a certain point, defined by the viscosity peak, granule swelling is so extensive that the resulting highly expanded structures are susceptible to mechanically-induced fragmentation under the stirring conditions used. With increased heating and holding at 95.degree. C. further reduction in viscosity is observed due to increased fragmentation of swollen granules. This general profile (FIG. 5) has previously always been found for native potato starch. In addition to the overall shape of the viscosity response in a viscoamylograph, a convenient quantitative measure is the temperature of initial viscosity development (onset). FIG. 2 shows a typical viscosity profile for starch (Kennedy & Cabalda, Chem. in Britain. November 1991, 1017-1019), during and after cooking, with a representation of the physical state of the starch granules at various points. The letters A, B, C and D correspond to the stages of viscosity onset (A), maximum viscosity (B), complete dispersion (C) and re-association of molecules (or retrogradation, D).
The properties of potato starch are useful in a variety of both food and non-food (paper, textiles, adhesives etc.) applications. However, for many applications, properties are not optimum and various chemical and physical modifications well known in the art are undertaken in order to improve useful properties. Two types of property manipulation which would be of use are firstly the controlled alteration of gelatinisation and pasting temperatures and, secondly, starches which do not suffer as much granular fragmentation during pasting as illustrated in FIG. 1. Currently the only ways of manipulating the gelatinisation and pasting temperatures of potato starch are by the inclusion of additives such as sugars, polyhydroxy compounds of salts (Evans and Haisman, Starke 34, 224-231, 1982) or by extensive physical or chemical pre-treatments (e.g. Stute, Starke 44, 205-214, 1992). The reduction of granule fragmentation during pasting can be achieved either by extensive physical pre-treatments (Stute, Starke 44, 205-214, 1992) or by chemical cross-linking. Such processes are inconvenient and inefficient. It is therefore desirable to obtain plants which produce starch which intrinsically possesses such advantageous properties.
Starch Biosynthesis
Starch consists of 2 major components: amylose, a linear polymer of alpha, 1-4 linked glucose units; and amylopectin, a branched polvmer consisting of an alpha, 1-4 linked glucan backbone with alpha, 1-6 linked branches. The key enzymes in starch biosynthesis are the starch synthases and starch branching enzyme [alpha-1,4-glucan: alpha-1,4-glucan 6-glucosyltransferase. EC 2.4.1.18]. Amylose is synthesized from adenosine 5'-(alpha-D-glucopyranosyl pyrophosphate), or "ADP-glucose", by a starch synthase which is associated with the starch granule: the so-called "granule bound starch synthase" (GBSS). Amylopectin is synthesized from ADP-glucose by the concerted action of a soluble starch synthase (SSS) and starch branching enzyme (SBE). SBE hydrolyses the linear alpha-1-4 glucan chain and rejoins the cleaved portion via an alpha-1-6 linkage to produce a branched structure. The activity of SBE is thus of crucial importance in determining the type, and hence properties, of starch synthesized within plant systems.
Starch Branching Enzyme
In most plant species, SBE occurs in multiple forms (e.g. maize kernels. Boyer & Preiss, Biochem. Biophys. Res. Commun. 80, 169-175 (1978); sorghum seed, Boyer, Phytochem. 24, 15-18 (1985); rice endosperm, Smyth. Plant Sci. 57, 1-8 (1988); pea embryo, Smith, Planta 175, 270-279 (1988)). However, in potato tuber, only a single form of SBE has so far been identified (Blennow & Johansson, Phytochem. 30, 437-444 (1991)).
Endosperm of maize contains three forms of SBE, namely SBE I, SBE IIa and SBE IIb. The "amylose extender" (ae) mutation causes a large reduction of SBE activity and in particular loss of SBE IIb. This reduction in SBE activity results in a higher ratio of amylose to amylopectin in endosperm starch compared to normal maize (Boyer & Preiss, Biochem. Biophys. Res. Commun. 80, 169-175 (1978)).
In pea embryos, 2 forms of SBE exist. The r (wrinkled) mutant of pea lacks SBE I activity and starch from this source has a higher ratio of amylose to amylopectin than normal peas [Smith, Planta 175, 270-279 (1988)].
In potato, amylose-free mutants have been obtained by X-ray irradiation (Hoverkamp-Hermelink et al., Theor. Appl. Genet. 75, 217-221, 1987) and by transformation with antisense-GBSS constructs (Visser et al., Mol. Gen. Genet. 225, 289-296, 1991). However, no high amylose mutants of potato exist and efforts to produce such via transformation with antisense SBE constructs have, hitherto, been unsuccessful (e.g. DE 41 04782A1). In respect of the latter, Wilmitzer et al., [Proceedings International Symposium on Plant Polymeric Carbohydrates, ed. Meuser. Manners & Siebel (1992) pp 33-39] have, using antisense SBE technology, produced tubers containing only 10-20% SBE activity of control tubers, but: "neither the amylose content of the starch in the tubers of these plants, nor the total starch content of the tubers, was altered" (p.39). Similarly, WO 92/11375 suggests the use of an anti-sense approach to alter the starch content of tubers, but there was no reduction to practice and no data showing success of the approach, which disclosure cannot therefore be considered as enabling.
The present inventors have been able to employ similar techniques to obtain plants with even lower levels of SBE activity than those described by Wilmitzer. Surprisingly, especially in view of Wilmitzer's results, the starch obtained from such plants has unexpected novel, commercially useful properties.